Chromatographic methods

ABSTRACT

The current invention comprises a method for the regeneration of a cation exchange chromatography column.

PRIORITY TO RELATED APPLICATION(S)

This application is a divisional of application Ser. No. 12/218,543,filed Jul. 16, 2008, now U.S. Pat. No. 7,846,336 which claims thebenefit of European Patent Application No. 07013960.5, filed Jul. 17,2007, which are hereby incorporated by reference in their entirety.

The current invention is in the field of chromatographic separationmethods useful for the purification of polypeptides, especially ofPEGylated polypeptides.

BACKGROUND OF THE INVENTION

Proteins play an important role in today's medical portfolio. For humanapplication every therapeutic protein has to meet distinct criteria. Toensure the safety of biopharmaceutical agents to humans by-productsaccumulating during the production process have to be removedespecially. To fulfill the regulatory specifications one or morepurification steps have to follow the manufacturing process. Among otherthings, purity, throughput, and yield play an important role indetermining an appropriate purification process.

Different methods are well established and widespread used for proteinpurification, such as affinity chromatography with microbial proteins(e.g., protein A or protein G affinity chromatography), ion exchangechromatography (e.g., cation exchange (sulfopropyl or carboxymethylresins), anion exchange (amino ethyl resins) and mixed-mode ionexchange), thiophilic adsorption (e.g., with beta-mercaptoethanol andother SH ligands), hydrophobic interaction or aromatic adsorptionchromatography (e.g., with phenyl-Sepharose, aza-arenophilic resins, orm-aminophenylboronic acid), metal chelate affinity chromatography (e.g.,with Ni(II)- and Cu(II)-affinity material), size exclusionchromatography, and electrophoretical methods (such as gelelectrophoresis, capillary electrophoresis) (Vijayalakshmi, M. A., Appl.Biochem. Biotech. 75 (1998) 93-102).

Conjugations are reported, for example, for polyethylene glycol (PEG)and Interleukin-6 (EP 0 442 724), for PEG and erythropoietin (WO01/02017), for chimeric molecules comprising Endostatin andimmunoglobulins (US 2005/008649), for secreted antibody based fusionproteins (US 2002/147311), for fusion polypeptides comprising albumin(US 2005/0100991; human serum albumin U.S. Pat. No. 5,876,969), forPEGylated polypeptides (US 2005/0114037), and for interferon fusions.

Necina, R., et al. (Biotechnol. Bioeng. 60 (1998) 689-698) reported thecapture of human monoclonal antibodies directly from cell culturesupernatants by ion exchange media exhibiting high charge density. In WO89/05157 a method is reported for the purification of productimmunoglobulins by directly subjecting the cell culture medium to acation exchange treatment. A one-step purification of monoclonal IgGantibodies from mouse ascites is described by Danielsson, A., et al., J.Immun. Meth. 115 (1988), 79-88. A method for purifying a polypeptide byion exchange chromatography is reported in WO 2004/024866 in which agradient wash is used to resolve a polypeptide of interest from one ormore contaminants. In EP 0 530 447 a process for purifying IgGmonoclonal antibodies by a combination of three chromatographic steps isreported. A facile purification of mono-PEGylated interleukin-1 receptorantagonist is reported by Yu, G., et al., in Process Biotechnol. 42(2007) 971-977. Wang et al. (Wang, H., et al., Peptides 26 (2005)1213-1218) reports the purification of hTFF3 expressed in E. coli by atwo step cation exchange chromatography. Yun et al. (Yun, Q., et al., J.Biotechnol. 118 (2005) 67-74) report the purification of PEGylatedrhG-CSF by two consecutive ion-exchange chromatography steps.

SUMMARY OF THE INVENTION

One aspect of the current invention is a method for the regeneration ofa cation exchange chromatography column after the elution of compoundsof interest comprising the following steps in this order:

eluting adsorbed polypeptides from the column with an aqueous bufferedsolution comprising sodium chloride at a concentration of at least 500mM,

flushing the column with purified water,

applying a 0.5 M sodium hydroxide solution to the column,

flushing the column with purified water, applying a solution comprising0.5 M sodium dihydrogen phosphate and 1 M phosphoric acid to the column,

flushing the column with purified water,

applying a 0.5 M sodium hydroxide solution to the column for at least 4hours, and

regenerating the cation exchange column by flushing the column withpurified water.

DETAILED DESCRIPTION OF THE INVENTION

The current invention provides in a first aspect, a method for theregeneration of a cation exchange chromatography column after theelution of compounds of interest comprising the following steps:

removing any residual bound polypeptides from the cation exchange columnwith a aqueous buffered solution comprising sodium chloride, followed by

flushing the cation exchange column with purified water, followed by

applying a sodium hydroxide solution to the cation exchange column,followed by

flushing the column with purified water, followed by

applying a solution comprising sodium dihydrogen phosphate andphosphoric acid to the cation exchange column, followed by

flushing the cation exchange column with purified water, followed by

applying a 0.5 M sodium hydroxide solution to the cation exchange columnfor at least 4 hours, and

regenerating the cation exchange column by flushing the cation exchangecolumn with purified water.

The term “purified water” as used within this application denotes waterfor injection according to the US Pharmacopeia.

The term “ion exchange material” as used within this application denotesan immobile, high molecular weight matrix that carries covalently boundcharged substituents that are used as stationary phase in ion exchangechromatography. For overall charge neutrality nor covalently boundcounter ions are bound thereto. The “ion exchange material” has theability to exchange its nor covalently bound counter ions for similarlycharged ions of the surrounding solution. Depending on the charge of itsexchangeable counter ions the “ion exchange resin” is referred to as acation exchange resin or as an anion exchange resin. Depending on thenature of the charged group (substituent), the “ion exchange resin” isreferred to in the case of cation exchange resins, as sulfonic acidresin (S), sulfopropyl resin (SP), or carboxymethyl resin (CM).Depending on the chemical nature of the charged group/substituent the“ion exchange resin” can also be classified as a strong or weak ionexchange resin, depending on the strength of the covalently boundcharged substituent. For example, strong cation exchange resins have asulfonic acid group, preferably a sulfopropyl group, as the chargedsubstituent, while weak cation exchange resins have a carboxylic group,preferably a carboxymethyl group, as the charged substituent, and weakanion exchange resins have a diethylaminoethyl group as the chargedsubstituent. In one embodiment, the cation exchange chromatographycolumn contains a sulfopropyl cation exchange resin, i.e., it is asulfopropyl cation exchange chromatography column.

Different types of ion exchange materials, i.e., stationary phases, areavailable under different names and from a multitude of companies suchas e.g., cation exchange materials Bio-Rex® (e.g., type 70), Chelex®(e.g., type 100), Macro-Prep® (e.g., type CM, High S, 25 S), AG® (e.g.,type 50W, MP) all available from BioRad Laboratories, WCX 2 availablefrom Ciphergen, Dowex® MAC-3 available from Dow chemical company,Mustang C and Mustang S available from Pall Corporation, Cellulose CM(e.g., type 23, 52), hyper-D, partisphere available from Whatman plc.,Amberlite® IRC (e.g., type 76, 747, 748), Amberlite® GT 73, Toyopearl®(e.g., type SP, CM, 650M) all available from Tosoh Bioscience GmbH, CM1500 and CM 3000 available from BioChrom Labs, SP-Sepharose™,CM-Sepharose™ available from GE Healthcare, Poros resins available fromPerSeptive Biosystems, Asahipak ES (e.g., type 502C), CXpak P, IEC CM(e.g., type 825, 2825, 5025, LG), IEC SP (e.g., type 420N, 825), IEC QA(e.g., type LG, 825) available from Shoko America Inc., 50W cationexchange resin available from Eichrom Technologies Inc. In oneembodiment the cation exchange material is a strong cation exchangematerial such as Macro-Prep® High S or 25S, or MacroCap SP, orToyopearl® SP 650M, or Source S, or SP Sepharose, or POLYCAT A.Exemplary anion exchange materials are Dowex® 1 available from Dowchemical company, AG® (e.g., type 1, 2, 4), Bio-Rex® 5, DEAE Bio-Gel 1,Macro-Prep® DEAE all available from BioRad Laboratories, anion exchangeresin type 1 available from Eichrom Technologies Inc., Source Q, ANXSepharose 4, DEAE Sepharose (e.g., type CL-6B, FF), Q Sepharose, CaptoQ, Capto S all available from GE Healthcare, AX-300 available fromPerkinElmer, Asahipak ES-502C, AXpak WA (e.g., type 624, G), IEC DEAEall available from Shoko America Inc., Amberlite® IRA-96, Toyopearl®DEAE, TSKgel DEAE all available from Tosoh Bioscience GmbH, Mustang Qavailable from Pall Corporation.

The term “flushing” as used within this application denotes the washingof a column with two or more column volumes of a specified solution.

The term “same type of cation exchange material” denotes two consecutiveion exchange chromatography steps which are performed by employing anidentical cation exchange material. This means that the consecutivecation exchange chromatography steps are carried out by using either afirst portion of the cation exchange material for the first cationexchange chromatography step and by using the second portion of the samecation exchange material for the second cation exchange chromatographyor by using the same cation exchange material for both cation exchangechromatography steps.

The terms “step elution” and “step elution method”, which are usedinterchangeably within this application, denote a method wherein theconcentration of a substance causing elution, i.e., the dissolution of abound compound from a material, is raised or lowered at once, i.e.,directly from one value/level to the next value/level. In this “stepelution” one or more conditions, for example the pH, the ionic strength,concentration of a salt, and/or the flow of a chromatography, is/arechanged all at once from a first, e.g., starting, value to a second,e.g., final, value, i.e., the conditions are changed incrementally,i.e., stepwise, in contrast to a linear change. In the “step elutionmethod” a new fraction is collected after each increase in the ionicstrength. This fraction contains the compounds recovered from the ionexchange material with a corresponding increase in ionic strength. Aftereach increase the conditions are maintained until the next step in theelution method is carried out.

The terms “continuous elution” and “continuous elution method”, whichare used interchangeably within this application, denote a methodwherein the concentration of a substance causing elution, i.e., thedissolution of a bound compound from a material, is raised or loweredcontinuously, i.e., the concentration is changed by a sequence of smallsteps each of which is not larger than a change of 2%, preferably of 1%,of the concentration of the substance causing elution. In this“continuous elution” one or more conditions, for example the pH, theionic strength, concentration of a salt, and/or the flow of achromatography, may be changed linearly or exponentially orasymptotically. Preferably the change is linear.

The term “applying to” and grammatical equivalents thereof as usedwithin this application denotes a partial step of a purification methodin which a solution containing, e.g., a substance of interest to bepurified, is brought in contact with a stationary phase. This denotesthat a) the solution is added to a chromatographic device in which thestationary phase is located, or b) that a stationary phase is added tothe solution. In case a) the solution containing, e.g., the substance ofinterest to be purified, passes through the stationary phase, allowingfor an interaction between the stationary phase and the substances insolution. Depending on the conditions, such as pH, conductivity, saltconcentration, temperature, and/or flow rate, some substances in thesolution are bound to the stationary phase and, thus, are removed fromthe solution. Other substances remain in solution or are desorbed fromthe stationary phase. The substances in solution can be found in theflow-through. The “flow-through” denotes the solution obtained after thepassage through the chromatographic device, which may either be theapplied solution containing the substance of interest or the buffer,which is used to flush the column or to cause elution of one or moresubstances bound to the stationary phase. In one embodiment thechromatographic device is a column, or a cassette. The substance ofinterest can be recovered from the solution after the purification stepby methods familiar to a person of skill in the art, such asprecipitation, salting out, ultrafiltration, diafiltration,lyophilization, affinity chromatography, or solvent volume reduction, toobtain the substance of interest in substantially homogeneous form. Incase b) the stationary phase is added, e.g., as a solid, to the solutioncontaining, e.g., the substance of interest to be purified, allowing foran interaction between the stationary phase and the substances insolution. After the interaction the stationary phase is removed, e.g.,by filtration, whereby the substance of interest is either bound to thestationary phase and removed therewith from the solution or remainsunbound to the stationary phase and remains in the solution.

The term “under conditions suitable for binding” and grammaticalequivalents thereof as used within this application denotes that asubstance of interest, e.g., PEGylated erythropoietin, binds to astationary phase, e.g., an ion exchange material when brought in contactwith it. This does not necessarily mean that 100% of the substance ofinterest is bound but that essentially 100% of the substance of interestis bound, i.e., at least 50% of the substance of interest is bound,preferably at least 75% of the substance of interest, more preferably atleast 85% of the substance of interest, and even more preferably morethan 95% of the substance of interest is bound to the stationary phase.

The term “buffered” as used within this application refers to a solutionin which changes of pH due to the addition or release of acidic or basicsubstances is leveled by a buffer substance. Any buffer substanceresulting in such an effect can be used. In one embodiment,pharmaceutically acceptable buffer substances are used, such asphosphoric acid or salts thereof, acetic acid or salts thereof, citricacid or salts thereof, morpholine, 2-(N-morpholino) ethanesulfonic acidor salts thereof, histidine or salts thereof, glycine or salts thereof,or tris (hydroxymethyl) aminomethane (TRIS) or salts thereof. Inpreferred embodiments, phosphoric acid or salts thereof, acetic acid orsalts thereof, citric acid or salts thereof, histidine or salts thereofare used. Optionally, the buffered solution may comprise one or moreadditional salts, such as sodium chloride, sodium sulphate, potassiumchloride, potassium sulfate, sodium citrate, or potassium citrate.

General chromatographic methods and their use are known to a personskilled in the art. See for example, Chromatography, 5^(th) edition,Part A: Fundamentals and Techniques, Heftmann (ed) Elsevier SciencePublishing Company 1992 Chromatography 5^(th) ed 51 A 1992; AdvancedChromatographic and Electromigration Methods in Biosciences, Deyl, Z.(ed.), Elsevier Science BV, Amsterdam, The Netherlands, (1998);Chromatography Today, Poole, C. F., and Poole, S. K., Elsevier SciencePublishing Company, New York, (1991); Scopes, Protein Purification:Principles and Practice (1982); Sambrook, J., et al. (ed), MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; or Current Protocolsin Molecular Biology, Ausubel, F. M., et al. (eds), John Wiley & Sons,Inc., New York.

The PEGylation of erythropoietin normally results in a mixture ofdifferent compounds, such as poly-PEGylated erythropoietin,mono-PEGylated erythropoietin, not-PEGylated erythropoietin, hydrolysisproducts of the activated PEG ester, as well as hydrolysis products ofthe erythropoietin itself. In order to obtain a mono-PEGylatederythropoietin in substantially homogeneous form these substances haveto be separated and the compound of interest has to be purified.

Therefore, it is the second aspect of the current invention to provide amethod for obtaining a mono-PEGylated erythropoietin in substantiallyhomogenous form comprising the following steps:

-   a) PEGylating erythropoietin using an activated PEGylating reagent    of a molecular weight of from 20 kDa to 40 kDa,-   b) purifying the PEGylated erythropoietin obtained in step a) with    two consecutive cation exchange chromatography steps, wherein the    first and second cation exchange chromatography steps employ the    same type of cation exchange material,-   c) recovering the mono-PEGylated erythropoietin from the second    cation exchange chromatography column in a substantially homogeneous    form,-   d) regenerating the cation exchange chromatography column by a    method according to the invention.

This method is especially useful for the purification of PEGylatedrecombinant polypeptides, which are glycosylated, i.e., which have beenproduced by a mammalian cell, preferably a CHO cell, HEK293 cell, BHKcell, Per.C6® cell, or HeLa cell and are afterwards chemicallyPEGylated. In one embodiment the regeneration of a cation exchangechromatography column comprises the following steps:

removing bound polypeptides from the cation exchange column with anaqueous buffered solution comprising sodium chloride,

flushing the column with purified water, preferably with at least twocolumn volumes,

applying a sodium hydroxide solution to the column, preferably at leasttwo column volumes,

flushing the column with purified water, preferably with at least twocolumn volumes,

applying a solution comprising sodium dihydrogen phosphate andphosphoric acid to the column, preferably at least three column volumes,

flushing the column with purified water, preferably with at least twocolumn volumes,

applying a 0.5 M sodium hydroxide solution to the column for at least 4hours, preferably for 4 hours, and

regenerating the cation exchange column by flushing the column withpurified water, preferably with at least two column volumes.

In the first step of the method, erythropoietin is PEGylated. The poly(ethylene glycol) (PEG) polymer molecules used in the PEGylationreaction have a molecular weight of about 20 kDa to 40 kDa (by“molecular weight” as used here there is to be understood the meanmolecular weight of the PEG because PEG as a polymeric compound is notobtained with a defined molecular weight but in fact has a molecularweight distribution; the term “about” indicates that in said PEGpreparations, some molecules will weigh more and some less than theindicated molecular weight, i.e the term “about” refers to a molecularweight distribution in which 95% of the PEG molecules have a molecularweight within +/−10% of the indicated molecular weight. For example, amolecular weight of 30 kDa denotes a range of from 27 kDa to 33 kDa).

The term “erythropoietin” refers to a protein having the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a protein or polypeptidesubstantially homologous thereto, whose biological properties relate tothe stimulation of red blood cell production and the stimulation of thedivision and differentiation of committed erythroid progenitors in thebone marrow. Recombinant erythropoietin may be prepared via expressionin eukaryotic cells, for example in CHO cells, or BHK cells, or HeLacells by recombinant DNA technology or by endogenous gene activation,i.e., the erythropoietin glycoprotein is expressed by endogenous geneactivation. See, for example U.S. Pat. No. 5,733,761, U.S. Pat. No.5,641,670, U.S. Pat. No. 5,733,746, WO 93/09222, WO 94/12650, WO95/31560, WO 90/11354, WO 91/06667, and WO 91/09955. In one embodimentthe erythropoietin according to the invention is based on the sequenceof human EPO. In a preferred embodiment the human erythropoietin has theamino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, morepreferably the human erythropoietin has the amino acid sequence set outin SEQ ID NO: 1. The term “erythropoietin” also denotes variants of theprotein of SEQ ID NO: 1 or SEQ ID NO: 2, in which one or more amino acidresidues have been changed, deleted, or inserted, and which has the samebiological activity as the non-modified protein, such as e.g., reportedin EP 1 064 951, or U.S. Pat. No. 6,583,272. A variant may have theamino acid sequence of human erythropoietin having from 1 to 6additional sites for glycosylation. The specific activity of PEGylatederythropoietin can be determined by various assays known in the art. Thebiological activity of the purified PEGylated erythropoietin of thisinvention are such that administration of the protein by injection tohuman patients results in bone marrow cells increasing production ofreticulocytes and red blood cells compared to non-injected or controlgroups of subjects. The biological activity of the PEGylatederythropoietin obtained and purified in accordance with this inventioncan be tested by methods according to Pharmeuropa Spec. IssueBiologicals BRP Erythropoietin Bio 97-2 (1997) 31-48.

“PEG” or “PEG group” according to the invention means a residuecontaining poly (ethylene glycol) as an essential part. Such a PEG cancontain further chemical groups, which are necessary for bindingreactions, which results from the chemical synthesis of the molecule, orwhich are spacers for optimal distance of parts of the molecule. Thesefurther chemical groups are not used for the calculation of themolecular weight of the PEG polymer molecule. In addition, such a PEGcan consist of one or more PEG side-chains which are linked together.PEGs with more than one PEG chain are called multiarmed or branchedPEGs. Branched PEGs can be prepared, for example, by the addition ofpolyethylene oxide to various polyols, including glycerol,pentaerythriol, and sorbitol. Branched PEG are described in, forexample, EP 0 473 084, U.S. Pat. No. 5,932,462. Linear PEG moleculeswith a molecular weight of 20-35 kDa molecules are used in oneembodiment and branched PEG polymers with a molecular weight of morethan 35 kDa, especially with 40 kDa, are used in another embodiment. A40 kDa, a two-armed PEG is particularly preferred.

The term “PEGylation” means a covalent linkage of a poly (ethyleneglycol) residue at the N-terminus of the polypeptide and/or an internallysine residue. PEGylation of proteins is widely known in the state ofthe art and is reviewed by, for example, Veronese, F. M., Biomaterials22 (2001) 405-417. PEG can be linked using different functional groupsand poly (ethylene glycol)s with different molecular weight, linear andbranched PEGs as well as different linking groups (see also Francis, G.E., et al., Int. J. Hematol. 68 (1998) 1-18; Delgado, C., et al., Crit.Rev. Ther. Drug Carrier Systems 9 (1992) 249-304) can be used.PEGylation of erythropoietin can be performed in aqueous solution withPEGylation reagents as described, for example, in WO 00/44785, in oneembodiment, using NHS-activated linear or branched PEG molecules with amolecular weight between 5 kDa and 40 kDa. PEGylation can also beperformed at the solid phase according to Lu, Y., et al., ReactivePolymers 22 (1994) 221-229. Not randomly, N-terminally PEGylatedpolypeptide can also be produced according to WO 94/01451.

Such methods result in an erythropoietin which is PEGylated at one ormore ε-amino groups of lysine residues and/or at the N-terminal aminogroup. Selective PEGylation at the N-terminal amino acid can beperformed according to Felix, A. M., et al., ACS Symp. Ser. 680(Poly(ethylene glycol)) (1997) 218-238. Selective N-terminal PEGylationcan be achieved during solid-phase synthesis by coupling of aN^(α)-PEGylated amino acid derivative to the N−1 terminal amino acid ofthe peptide chain. Side chain PEGylation can be performed duringsolid-phase synthesis by coupling of N^(ε)-PEGylated lysine derivativesto the growing chain. Combined N-terminal and side chain PEGylation isfeasible either as described above within solid-phase synthesis or bysolution phase synthesis by applying activated PEG reagents to an aminodeprotected peptide.

Suitable PEG derivatives are activated PEG molecules with in oneembodiment an average molecular weight of from about 5 to about 40 kDa,in a preferred embodiment of from about 20 to about 40 kDa, and in amore preferred embodiment of about 30 kDa to about 35 kDa. The PEGderivatives can be linear or branched PEGs. A wide variety of PEGderivatives suitable for use in the preparation of PEG-protein andPEG-peptide conjugates can be obtained from Shearwater Polymers(Huntsville, Ala., U.S.A.).

Activated PEG derivatives are known in the art and are described in, forexample, Morpurgo, M., et al., J. Bioconjug. Chem. 7 (1996) 363-368, forPEG-vinylsulfone. Linear chain and branched chain PEG species aresuitable for the preparation of the PEGylated fragments. Examples ofreactive PEG reagents are iodo-acetyl-methoxy-PEG, ormethoxy-PEG-vinylsulfone (m is in one embodiment an integer of fromabout 450 to about 900 and R is lower alkyl, linear or branched, havingone to six carbon atoms such as methyl, ethyl, isopropyl, etc. wherebymethyl is preferred).

The use of these iodo-activated substances is known in the art anddescribed e.g., by Hermanson, G. T., in Bioconjugate Techniques,Academic Press, San Diego (1996) p. 147-148.

In one embodiment the PEG species is an activated PEG ester, e.g.,N-hydroxysuccinimidyl propionate, or N-hydroxysuccinimidyl butanoate, orN-hydroxysuccinimides such as PEG-NHS (Monfardini, C., et al.,Bioconjugate Chem. 6 (1995) 62-69). In one embodiment the PEG isactivated by N-hydroxysuccinimide ester

using alkoxy-PEG-N-hydroxysuccinimide, such asmethoxy-PEG-N-hydroxysuccinimide (MW 30000; Shearwater Polymers, Inc.),wherein R and m are as defined above. In one embodiment the PEG speciesis the N-hydroxysuccinimidyl ester of methoxy poly (ethyleneglycol)-butyric acid. The term “alkoxy” refers to an alkyl ether groupin which the term ‘alkyl’ means a straight-chain or branched-chain alkylgroup containing a maximum of four carbon atoms, such as methoxy,ethoxy, n-propoxy and the like, preferably methoxy.

The term “substantially homogeneous form” as used within thisapplication denotes that the PEGylated erythropoietin obtained,contained, or used are those having a defined number of PEG groupattached. In one embodiment the PEGylated erythropoietin is amono-PEGylated erythropoietin. The preparation may contain unreacted(i.e., PEG group lacking) erythropoietin, poly-PEGylated erythropoietin,as well as fragments of the polypeptide generated during the PEGylationreaction. The term “substantially homogeneous form” denotes that apreparation of a mono-PEGylated erythropoietin contains at least 50%(w/w) of the mono-PEGylated erythropoietin, at least 75% of themono-PEGylated erythropoietin, at least 90% of the mono-PEGylatederythropoietin, or more than 95% of the mono-PEGylated erythropoietin.The percent values are based on the area-% of the chromatogramcorresponding to the cation exchange chromatography from which themono-PEGylated erythropoietin is obtained.

The present invention describes a method for the purification of amono-PEGylated erythropoietin in order to obtain a substantiallyhomogeneous form of a mono-PEGylated erythropoietin. It has surprisinglybeen found that the combination of two consecutive cation exchangechromatography steps both of which employ the same type of cationexchange material provides a substantially homogeneous form of amono-PEGylated erythropoietin. Therefore the current invention providesa method for the purification of a mono-PEGylated erythropoietincomprising the steps of providing a solution comprising mono-, poly-,and not-PEGylated erythropoietin, performing two consecutive cationexchange chromatography steps, recovering the purified mono-PEGylatederythropoietin in the second cation exchange chromatography step,wherein the same type of cation exchange material is used in both cationexchange chromatography steps, and regenerating the cation exchangechromatography column by a method according to the first aspect of thecurrent invention.

The recovery of the purified mono-PEGylated erythropoietin in the secondcation exchange chromatography step is carried out by eluting themono-PEGylated erythropoietin from the second cation exchangechromatography material. In one embodiment of the invention, the twocation exchange chromatography steps differ in the elution methodemployed. The first cation exchange chromatography step is performed inone embodiment, as a step elution method, i.e., the ionic strength ofthe used buffer is increased stepwise, i.e., at once, from one ionicstrength value to the next ionic strength value. The step elution methodis performed in one embodiment as a three step elution method. In thefirst step mainly poly-PEGylated erythropoietin is eluted from thecation exchange chromatography column. The second increase in ionicstrength basically elutes the mono-PEGylated erythropoietin with apurity of more than 60% based on the area of the correspondingsize-exclusion chromatogram (area-%). The third increase in ionicstrength elutes mainly the remaining not-PEGylated erythropoietin fromthe column.

The second cation exchange chromatography step is performed in oneembodiment as a continuous elution method, i.e., the ionic strength ofthe buffer is increased continuously. The eluted fractions containingthe mono-PEGylated erythropoietin are combined in order to obtain amono-PEGylated erythropoietin in substantially homogeneous form,containing in one embodiment less than 0.5% of low molecular weightforms based on the area of the corresponding chromatogram. The buffer isin one embodiment present in a concentration of from 10 mM to 250 mM,preferably of from 50 mM to 150 mM, more preferably at about 100 mM.

Therefore, in the method according to the invention the two consecutivecation exchange chromatography steps are the following:

a) applying an aqueous, buffered solution comprising a mixture of mono-,poly-, and non-PEGylated erythropoietin to a first cation exchangechromatography column under conditions suitable for binding of saidmono-PEGylated erythropoietin to the cation exchange material containedin said first column,b) recovering a mono-PEGylated erythropoietin from the first cationexchange chromatography column by a step elution method with a stepwiseincrease of the ionic strength of the through flowing buffer, whereinthe relative content of mono-PEGylated erythropoietin is increasedcompared to the applied mixture of step a),c) applying the recovered mono-PEGylated erythropoietin from step b) toa second cation exchange chromatography column under conditions suitablefor binding of said erythropoietin to the cation exchange materialcontained in said second column, whereby the cation exchange materialcontained in said second column is of the same type as the cationexchange material in the first column,d) recovering the purified mono-PEGylated erythropoietin in asubstantially homogeneous form from said second cation exchangechromatography column by a continuous elution method with a continuousincrease of the ionic strength of the through flowing buffer.

The PEGylation of a polypeptide does normally not provide the PEGylationproduct in homogeneous form. Rather, it is obtained as a mixture ofmono-PEGylated, poly-PEGylated, and non-PEGylated product. Therefore thesolution of the PEGylated erythropoietin applied in step a) of themethod is a mixture of mono-, poly-, and non-PEGylated erythropoietinand low molecular weight forms or fragments in an aqueous buffer. Therelative content of the different substances is determined by sizeexclusion chromatography (SE-HPLC). The sum of the area of thecorrelated peaks, i.e., the area under the peaks, in the size exclusionchromatogram is the total area of the size-exclusion chromatogram. Thefraction of a single peak is given as area-%, i.e., as relative areafraction of the total area of the chromatogram.

General chromatographic methods, their use, and the related terms areknown to a person skilled in the art. See for example, Chromatography,5^(th) edition, Part A: Fundamentals and Techniques, Hellmann (ed.),Elsevier Science Publishing Company, ed., Chromatography 5^(th) ed., 51A (1992) and other related textbooks. During the chromatography a bufferis flowing through the cation exchange chromatography column. This“through flowing buffer” is adjusted according to the requirements ofthe steps of the chromatography method. It transports the substance ofinterest to (applying) and from (eluting) the chromatographic material.

In the first cation exchange chromatography step the mixture ofmono-PEGylated, poly-PEGylated, and not-PEGylated erythropoietin isapplied at a protein concentration of about 1 mg/ml to the first cationexchange chromatography column in an aqueous solution buffered withabout 100 mM potassium phosphate at about pH 3.0. The term “about” asused within the current application denotes a range of 10% around thegiven value, i.e., ±10%. Prior to and after the application the firstcolumn is washed with the same buffer solution. For the first step inthe step elution method the buffer is changed to a buffer with about 100mM potassium phosphate, about 90 mM sodium chloride at about pH 3.0. Inusing this buffer hydrolyzed PEG reagent, i.e., the correspondingPEGylated carbonic acid, unreacted coupling reagent, and poly-PEGylatederythropoietin are eluted from the cation exchange chromatographycolumn. For the second step in the three step elution method the bufferis changed to a buffer with about 100 mM potassium phosphate, about 250mM sodium chloride at about pH 3.0. In this step the mono-PEGylatederythropoietin is recovered from the first cation exchangechromatography column. The collected through flowing buffer of thiselution step is diluted approximately 1:5 to 1:8 with purified water.After the first cation exchange chromatography step the recoveredmono-PEGylated erythropoietin is free of free PEG.

The collected, through flowing buffer of the second step of the firstcation exchange chromatography contains the mono-PEGylatederythropoietin in an increased relative content, i.e the fraction byweight or by area-% (in the chromatogram of a size exclusionchromatography of the collected through flowing buffer of the secondstep) of the mono-PEGylated erythropoietin has increased when comparedto prior to the first cation exchange chromatography step. In oneembodiment, the relative content of mono-PEGylated erythropoietin is atleast 60 area-%. In a preferred embodiment, the relative content ofmono-PEGylated erythropoietin is at least 80 area-%.

For further purification of the mono-PEGylated erythropoietin a secondcation exchange chromatography step is performed. For the second cationexchange chromatography the collected and diluted through flowing bufferof the second elution step is adjusted to a potassium phosphateconcentration of about 100 mM and to a pH of about pH 3.0 and is appliedto a second cation exchange chromatography column containing a cationexchange material of the same type as the first cation exchangechromatography column. In one embodiment, the second cation exchangecolumn and the cation exchange material contained therein are the sameas in the first cation exchange chromatography step. The mono-PEGylatederythropoietin is recovered from the second cation exchangechromatography column by applying a linear gradient starting withpotassium phosphate buffer at a concentration of about 100 mM with about50 mM sodium chloride at about pH 3.0 and ending with a potassiumphosphate buffer of a concentration of about 100 mM with about 500 mMsodium chloride at about pH 3.0. The change in the sodium chlorideconcentration is linear over ten column volumes. The through flowingbuffer is fractioned and each fraction is diluted with 1 M dipotassiumhydrogen phosphate to increase the pH value to about pH 6 to 8.

After the second cation exchange chromatography step the mono-PEGylatederythropoietin is obtained in substantially homogeneous form, preferablywith a purity of at least 95% by area.

A person of skill in the art is familiar with the technology of ionexchange chromatography. In the step leading to recovery of thepolypeptide bound to the cation exchange material the ionic strength,i.e., the conductivity, of the buffer/solution passing through the ionexchange column is increased. This can be accomplished either by anincreased buffer salt concentration or by the addition of other salts,so called elution salts, to the buffer solution. Depending on theelution method the buffer/salt concentration is increased at once (stepelution method) or continuously (continuous elution method) by thefractional addition of a concentrated buffer or elution salt solution.In one embodiment the elution salt is sodium citrate, sodium chloride,sodium sulphate, sodium phosphate, potassium chloride, potassiumsulfate, potassium phosphate, or other salts of citric acid orphosphoric acid, or any mixture of these components. In a preferredembodiment the elution salt is sodium citrate, sodium chloride,potassium chloride, or mixtures thereof.

In one embodiment of the invention, the cation exchange material is astrong cation exchange material. In a preferred embodiment, there isToyopearl® SP 650 M. In another preferred embodiment, it is asulfopropyl cation exchange material. The concentration of the salt,causing the elution, is in one embodiment in the range of from 5 mM to500 mM. In a preferred embodiment, the concentrated range from 5 mM to400 mM, and in an especially preferred embodiment, from 5 mM to 250 mM.In another embodiment of the invention, the salt causing the elution forexample citric acid or salts thereof or phosphoric acid or salts thereofis also used as buffer substance

The mono-PEGylated erythropoietin may be used in pharmaceuticalcompositions suitable for injection with a pharmaceutically acceptablecarrier or vehicle by methods known in the art. For example, appropriatecompositions have been described in WO 97/09996, WO 97/40850, WO98/58660, and WO 99/07401. Among the preferred, pharmaceuticallyacceptable carriers for formulating the products of the invention arehuman serum albumin, human plasma proteins, etc. The compounds of thepresent invention may be formulated in 10 mM sodium/potassium phosphatebuffer at pH 7 containing a tonicity agent, e.g., 132 mM sodiumchloride. Optionally the pharmaceutical composition may contain apreservative. The pharmaceutical composition may contain differentamounts of mono-PEGylated erythropoietin, e.g., 10-1000 μg/ml, e.g., 50μg or 400 μg.

Administration of the erythropoietin glycoprotein products of thepresent invention results in red blood cell formation in humans.Therefore, administration of the mono-PEGylated erythropoietinglycoprotein product replenishes this erythropoietin protein which isimportant in the production of red blood cells. The pharmaceuticalcompositions containing the mono-PEGylated erythropoietin glycoproteinproducts may be formulated at a strength effective for administration byvarious means to a human patient experiencing blood disorderscharacterized by low or defective red blood cell production, eitheralone or as part of a condition or disease. The pharmaceuticalcompositions may be administered by injection such as by subcutaneous orintravenous injection. Average quantities of the mono-PEGylatederythropoietin glycoprotein product may vary. The exact amount ofconjugate is a matter of preference subject to factors such as the exacttype of condition being treated, the condition of the patient beingtreated, as well as the other ingredients in the composition. Forexample, 0.01 to 10 μg per kg body weight, preferably 0.1 to 1 μg per kgbody weight, may be administered e.g. once weekly.

It has surprisingly been found that a cation exchange chromatographycolumn can be regenerated with a method according to the inventionwithout a considerable decline in the separation efficiency. It has beenshown that, using a regeneration method according to the invention, acation exchange chromatography column can be used for at least 40separation cycles, in one embodiment for at least 50 separation cycles,in a further embodiment for at least 60 separation cycles without aconsiderable decline in the separation efficiency (see FIG. 1) and yield(see FIG. 2). The term “separation cycle” as used within thisapplication denotes the sequence i) equilibration of the column, ii)application of the solution to be separated on the column, iii) washingthe column, iv) recovering the adsorbed compounds from the column, v)washing the column, vi) regenerating the column. It has also been foundthat with the regeneration method according to the invention not onlycan a decline in the separation efficiency be avoided but a decline inthe loading capacity can also be prevented (see FIG. 2).

The term “separation efficiency” as used within this application denotesthe ability of a cation exchange chromatography column to separate thecompounds of a solution. The term “without a considerable decline” asused within this application denotes that the cation exchangechromatography column provides the same, i.e., within in variation of+/−5%, in one embodiment within a variation of +/−2.5%, compoundseparation in consecutive chromatographies of a solution containing thesame compounds. The term “loading capacity” as used within thisapplication denotes the amount of a compound of interest that isrecovered from a cation exchange chromatography column.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Purity of mono-PEGylated erythropoietin in the through flowingbuffer pool of the first chromatography during cycle number validationof the regeneration process.

FIG. 2 Yield of mono-PEGylated erythropoietin in the through flowingbuffer pool of the first chromatography during cycle number validationof the regeneration process.

MATERIALS AND METHODS

SE-HPLC

SE-HPLC separates proteins according to their apparent molecular weight.Therefore, the method is able to detect the presence of mono-PEGylatederythropoietin, low molecular weight forms and fragments, poly-PEGylatedforms and higher aggregates of erythropoietin. The HPLC is equipped witha 220-nm detector and a Superose 6 HR column (dimensions 10×300 mm,Pharmacia Biotech, Cat-Nr: 17-0537-01) or a Superose 6 10/300 GL column(Pharmacia Biotech, Cat-Nr: 17-5172-01). The column is operated underisocratic conditions at room temperature, using a flow rate of about 0.4ml/min. The mobile phase buffer is a 50 mM sodium phosphate buffer with300 mM sodium chloride at pH 6.8. Dependent on the HPLC-system used, themethod can be performed with a sample application volume of either 100μL or 500 μL. The samples are diluted with the mobile phase buffer to aprotein concentration of about 0.5 mg/mL (100 μL load) or 0.1 mg/mL (500μL load). Samples with a protein concentration of less than 0.1 mg/mLcan be used undiluted. The eluted proteins are detected at a detectorwavelength of 220 nm.

RP-HPLC

Purity is analyzed by RP-HPLC, which separates mono-PEGylatederythropoietin from oligo forms and related substances. The assay isperformed on a Poroshell column using an acetonitrile/aqueous TFAgradient. The elution profile is monitored as UV absorbance at 220 nm.The percentage of mono-PEGylated erythropoietin and related substancesor oligo forms are calculated based upon the total peak area of theeluted proteins

Example 1 Purification of Mono-PEGylated Erythropoietin

Erythropoietin can be produced e.g. according to WO 01/87329, andpurified as reported in WO 96/135718. PEGylated erythropoietin can beproduced e.g. according to WO 03/029291.

a) First Chromatography on SP Toyopearl 650 M

The first chromatography step was performed on a sulfopropyl (SP) columnpacked with SP Toyopearl® 650M. The column was operated at roomtemperature. The maximum loading capacity of the first column wasdefined as 1.5 g protein per liter column volume (CV). The column wasequilibrated with a 100 mM potassium phosphate buffer at a pH of from2.9 to 3.1 (SP-A buffer). After the loading step, the column was washedand eluted with a series of potassium phosphate buffers containingincreasing amounts of NaCl. Free PEGylated carbonic acid, i.e.,hydrolyzed PEG reagent, and poly-PEGylated forms were removed in theflow-through and the subsequent washing step with SP-A buffer and 100 mMpotassium phosphate buffer, pH 2.9 to 3.1, containing 90 mM sodiumchloride (SP-B buffer), respectively. The mono-PEGylated erythropoietinwas eluted by applying a 100 mM potassium phosphate buffer, pH 2.9 to3.1, containing 250 mM sodium chloride (SP-C buffer), collected in avessel and directly diluted 1:5 with purified water. This collectedeluate is termed “SP eluate pool I”. The column was subsequently washedwith 100 mM potassium phosphate buffer, pH 2.9 to 3.1, containing 750 mMsodium chloride (SP-D buffer) to remove unreacted erythropoietin and thecolumn was regenerated.

b) Second Chromatography on SP Toyopearl 650 M

The second column was operated at room temperature. After equilibrationwith SP-A buffer the SP eluate pool I was applied to the column and thecolumn was thereafter washed with SP-A buffer. The mono-PEGylatederythropoietin was eluted by applying a linear gradient with a slope offrom 50 to 500 mM sodium chloride over ten column volumes, buffered with100 mM potassium phosphate buffer at pH 2.9 to 3.1. The product peak wasfractionated in up to 8 single fractions and each fraction was directlydiluted with 1 M dipotassium hydrogen phosphate to increase the pH to 6to 8. After the elution of mono-PEGylated erythropoietin was completed,the slope of the gradient can be increased leading to an immediatecolumn wash with 100 mM potassium phosphate pH 2.9 to 3.1 containing 500mM sodium chloride.

c) Regeneration of the SP Toyopearl 650 M Columns

The resins of both columns were regenerated in a sequence of sevensteps. The columns were flushed with purified water followed by a 0.5 Msodium hydroxide solution. The alkaline solution was displaced withpurified water followed by an acid wash (0.5 M sodium dihydrogenphosphate, 1 M phosphoric acid). After another purified water step, thecolumns were depyrogenated with 0.5 M sodium hydroxide for ≧4 hours.After caustic regeneration, the columns were washed with purified wateragain. The purified water (PW III) was produced by ultrafiltration. Thequality of PW III is equivalent to that of water for injection accordingto the US Pharmacopeia. Testing is performed according to thePharmocopeia referred to previously. During control runs performedaccording to the above outlined first chromatography step no residualprotein or PEG moieties could be detected in the respective throughflowing buffers. SDS-PAGE analysis of the resin, after 60 cycles, showedno residual protein or PEG moiety on the gel. Based on these data abatch-to-batch carryover of residual proteins and PEG moiety can beexcluded and thus the regeneration of the column is very effective (seealso FIG. 1). The determination of the yield obtained in eachchromatography showed no decline (see also FIG. 2).

TABLE 1 Summary of parameters for the column regeneration. ColumnParameters Column Flow Rate Step Buffer Solution Volumes [L/min] RinsePW III ≧2 1.6-2.1 Caustic 0.5 mol/L NaOH ≧2 1.6-2.1 column regenerationI Rinse PW III ≧2 1.6-2.1 Acid column 1 mol/L phosphoric acid ≧3 1.6-2.1regeneration 0.5 mol/L sodium dihydrogen phosphate Rinse PW III ≧21.6-2.1 Caustic 0.5 mol/L NaOH ≧3 n.a. column regeneration II Rinse PWIII ≧2 1.6-2.1

As shown in FIGS. 1 and 2, the purity and yield of mono-PEGylatederythropoietin in the through flowing buffer pool for the firstchromatography step for all cycles is clearly within the range of atleast 80% purity and at least 35% yield. In addition, no trend in purityof mono-PEGylated erythropoietin during the lifetime of the column canbe observed.

The invention claimed is:
 1. A method for obtaining a mono-PEGylatederythropoietin in substantially homogenous form comprising the followingsteps: a) PEGylating erythropoietin using an activated PEGylatingreagent of a molecular weight of from 20 kDa to 40 kDa to producePEGylated erythropoietin, b) purifying the PEGylated erythropoietinobtained in step a) with two consecutive cation exchange chromatographysteps, wherein the first and second cation exchange chromatography stepsemploy the same type of cation exchange material, c) recovering themono-PEGylated erythropoietin from the second cation exchangechromatography column in a substantially homogeneous form, d)regenerating the cation exchange chromatography column by: i) removingany bound polypeptide from the cation exchange column with an aqueousbuffered solution comprising sodium chloride, followed by ii) flushingthe column with purified water, followed by iii) applying a sodiumhydroxide solution to the column, followed by iv) flushing the columnwith purified water, followed by v) applying a solution comprisingsodium dihydrogen phosphate and phosphoric acid to the column, followedby vi) flushing the column with purified water, followed by vii)applying a 0.5 M sodium hydroxide solution to the column for at least 4hours, and followed by viii) regenerating the cation exchangechromatography column by flushing the column with purified water.
 2. Themethod according to claim 1, characterized in that said erythropoietinis human erythropoietin with an amino acid sequence of SEQ ID NO: 1 or2.
 3. The method according to claim 1, wherein said PEGylating reagentcomprises linear PEG having a molecular weight of from 20-35 kDa.
 4. Themethod according claim 1, wherein said PEGylating reagent comprises abranched PEG with a molecular weight of 40 kDa.
 5. The method accordingto claim 1, wherein said first cation exchange chromatography step isperformed as a step elution and said second cation exchangechromatography step is performed as a linear elution.